It has been found that a number of human diseases can be traced directly from genetic mutations. Various diagnostic assays have been developed to identify, detect and analyze unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from tissue or culture samples to determine the presence of physiological or pathological conditions. More particularly, the identification, detection and analysis of the unique DNA or RNA sequences or specific genes within the total DNA or RNA may indicate the presence of genetic disorders or cancer. In addition, infectious diseases can also be determined by the detection of DNA or RNA of the infectious agent.
Nucleic acid hybridization is used in many procedures of biotechnology and genetic engineering. Nucleic acid hybridization is a process in which single stranded nucleic acid pairs up with a complementary nucleotide sequence of another nucleic acid thereby forming a stable, double stranded DNA helix. Because of the requirement that hybridized nucleic acid strands have complementary nucleotide base sequences, hybridization processes are used to locate, detect and/or isolate specific nucleotide base sequences present on target nucleic acids.
Nucleic acid hybridization techniques have been applied to many procedures including but not limited to Southern blot detection of specific nucleic acid sequences (Southern, J. Mol. Biol., 98:503-17 (1975)); hybridization of polynucleotide primers in polymerase chain reaction to amplify the specific nucleic acid sequence. (U.S. Pat. Nos. 4,683,195 and 4,683,202 and 4,800,159 to Mullis et al; PCR Technology, Ehrlich, ed. Stockton press (1989); Faloona et al., Methods in Enzymol. 155:335-50 (1987): Polymerase Chain Reaction, Ehrlich, eds. Cold Spring Harbor Laboratories Press (1989); Saiki et al., Science, 239:487-491 (1988); Ehrlich et al., Science, 252:1643-1650 (1991)); library screening for cloning and manipulation of nucleic acid fragments into recombinant DNA cloning vectors (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (1982)); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987)); reverse blot hybridization using oligonucleotide with polythymidylate tails (Saiki et al., PNAS, 86:6230-6234 (1989)); reverse blot hybridization of PCR amplicons (Yong Zhang et al., Nuc. Acid Res., 19 (14):3929-3933 (1991)). All of the foregoing documents are hereby incorporated by reference.
For a method in which specificity is desired, hybridization between strands of nucleic acid that do not have complementary nucleotide base sequences should be avoided. Beck et al., Nuc. Acid Res., 16:9051 (1988) and Haqqi et al., Nuc. Acid Res., 16:1184 (1988).
Southern blot hybridization is a commonly used technique of nucleic acid hybridization and involves immobilizing a set of unknown target DNA molecules on a membrane and then immersing the membrane in a solution containing a labeled DNA probe molecule under conditions where the complementary molecules will anneal. Reverse blot hybridization is a modification of the Southern method. Specifically, instead of immobilizing unknown DNA, a set of well defined DNA probes is immobilized on a solid surface and the "unknown" labeled DNA is present in the liquid phase.
In instances where there are multiple possibilities of nucleic acid sequences which may be present, performing multiple assays by Southern blot hybridization is cumbersome and time consuming. However, reverse blot hybridization can be advantageously used in such instances because a large number of immobilized probes can be used with a single target sample. By decoding the hybridization pattern of the unknown DNA to positions of known sequence on a solid phase array, sequence information from several positions of the unknown DNA target can be obtained. Moreover, immobilized probe formats have great potential even in situations where the number of samples and probes are approximately equal because many filters can be prepared at one time and stored until needed. With the reverse blot, analysis of all the mutations with one filter is advantageous and saves considerable time and effort.
If a desired nucleic acid sequence is present in a small amount or the background caused by similar sequences in a sample is high, it can be difficult to obtain a reliable and sensitive detection of the targeted sequence. Amplification of the target nucleic acid by a process known as polymerase chain reaction (PCR) can be advantageously used in such cases.
K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202 which are incorporated herein by reference, describes polymerase chain reaction as a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing an excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of double stranded target sequence. To effect amplification, the mixture is denatured and the primers are then annealed to their complementary sequences with the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. There can be numerous "cycles" (i.e., each denaturation, primer annealing, and polymerase extension constitutes a "cycle") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other and, therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as "polymerase chain reaction". Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified".
Uracil-DNA Glycosylase (UDG) or Uracil-N-Glycosylase (UNG) is an enzyme that catalyzes the release of free uracil from single stranded and double stranded DNA of greater than 6 base-pairs. This enzyme has found important use in the prevention of PCR template carry over contamination. PCR reactions are run in the presence of 2'-deoxyuridine 5'- triphosphate (dUTP) instead of 2'-deoxythymidine 5'- triphosphate (dTTP). The resulting dUTP-amplicon can be analyzed in a normal manner. However, to prevent the transfer of the amplicon into other PCR reactions, UDG is added to hydrolyze the amplicon into fragments. Such fragments are unable to participate in the next round of PCR, thus arresting unwanted contamination.
Longo, et al., in Gene (1990) 93:125-128, describe the use of uracil-DNA glycosylase to control carry-over contamination in polymerase chain reactions. The method has two steps: (I) incorporating dUTP in all PCR products (by substituting dUTP for dTTP, or by incorporating uracil during synthesis of the oligodeoxyribonucleotide primer and (ii) treating all subsequent fully preassembled starting reactions with uracil-DNA glycosylase (UDG), followed by thermal inactivation of UDG.
Fraiser et al., U.S. Pat. No. 5,536,649 describes a method of inactivating amplicons in isothermal nucleic acid amplification such as strand displacement amplification (SDA) using UDG.
Rashtchian et al., Anal. Biochem., 206:91-97 (1992) describe the use of uracil-DNA glycosylase in mediating cloning of polymerase chain reaction amplified DNA.
Urdea, U.S. Pat. No. 4,775,619 describes a method for the detection of specific nucleotide sequences using hybridization. The duplex formation of the DNA and the probe affects the spatial relationship between a label and a support and the presence or absence of a particular sequence in a sample is determined by the amount of the label released into the medium. The technique provides a cleavage site between the label and the support through duplexing of the labeled probe and DNA sample. Urdea, U.S. Pat. No. 5,380,833 is a continuation-in-part of the foregoing patent, describes a solid phase hybridization assay where the sample is digested with restriction endonuclease into fragments.
Miyada et al., U.S. Pat. No. 5,525,717 is concerned with a support bound nucleotide probe specific for N. gonorrhoeae. The DNA sample is cleaved to obtain fragments that contain the target polynucleotide sequence.
Conventional hybridization methods of amplicons to reverse hybridization panels can be rather inefficient and require considerable time. This is apparently due to reassociation kinetics which are more favorable in the solution phase as opposed to the solid-phase. In addition, steric hindrance, secondary structure and concentration effects may also apply. Consequently, there is a need for improving the sensitivity of the detection of amplicons by reverse blot hybridization.